摘要
目的 :建立抗克伦特罗 (CL)单克隆抗体杂交瘤细胞系 ,制备CL单克隆抗体 ,并鉴定其免疫学特性。方法 :用重氮化法将半抗原CL偶联于载体蛋白BSA ,形成结合抗原BSA CL ;以BSA CL免疫BALB/C小鼠 ,应用杂交瘤技术建立分泌CL单克隆抗体细胞株 ;用体内诱生腹水法生产CL单抗 ,硫酸钠盐析法提纯。应用抗小鼠同种型抗体测定单抗的同种型 ,用竞争性ELISA和胶体金膜层析试验测定CL单抗的反应特异性和反应动力学。结果 :筛选出C 4G1、C 2H6、C 2D6和C 4C94株高敏感性和高特异性杂交瘤生产单抗 ,其单抗的间接ELISA效价分别为 5× 10 -5、3× 10 -4、6× 10 -4、2× 10 -5;同种型分别为IgG1/κ、IgG1/κ、IgG2a/κ、IgG2a/λ。C 4G1的亲和力常数K =1 6 8× 10 8L/mol,C 2H6的亲和力常数K =1 3× 10 8L/mol。经WestGold蛋白质印迹鉴定单抗与CL特异性结合。C 4G1单抗对CL的半数抑制浓度 (IC50 )为 7 94ng/ml,对沙丁胺醇 (Sb)的IC50 为 10 0 0ng/ml,对肾上腺素、去甲肾上腺素、异丙肾上腺素和来克多巴胺的IC50 均≥ 6 4 0 0ng/ml;C 4G1单抗与Sb的交叉反应性为0 79% ,与异丙肾上腺素的交叉反应性为 0 12 % ,而与肾上腺素、去甲肾上腺素、来克多巴胺、各类抗生素及多种维生素均无交叉反应性。结论
Objective:To generate hybridoma lines that secrete monoclonal antibodies(mAb)against clenbuterol(CL),a β-adrenergic agonist.Methods:CL-conjugated antigen (BSA-CL)were produced by coupling diazotized CL with Bovine Sera Albumin(BSA).The spleen cells of BALB/C mouse immunized by BSA-CL were fused with NS0 plasmacytoma cells using PEG4000,and selected cultrued with HAT and HT medium.Results:Four hybridoma cell lines of C4G1,C2H6,C2D6 and C4C9 were screened for specificity to CL,and cloned by limited dilution method for three times, which secrete high affinity monoclonal antibodies against CL with indirect ELISA titers of 5×10 -5 ,3×10 -4 ,6×10 -4 and 2×10 -5 in astices, respectively.Also, the four mAb have isotypes of IgG 1/κ、IgG 1/κ、IgG 2a /κ、IgG 2a /λ,respectively,and affinity constant is 1.68×10 8 L/mol in C4G1,1.3×10 8 L/mol in C2H6.CL mAb have specificity integrated with CL conjugated antigen that determined by WestGold protein blotting assay.The mAb of C4G1 showed good sensitivity with an IC 50 of 7.94 ng/ml to CL,1 000 ng/ml to salbuterol and more than 6 400 ng/ml to Norepinephrine,Epinephine,Isoprenaline and Ractopamine.And it generally had 0.79% cross-reactivity to Salbuterol,and showed little or no cross-reactivity to Norpinephrine,Epinephine,Isoprenaline,Ractopamine,antibiotics and multi-vitamin added in animal feed.Conclusion:CL conjugated antigen has been generated successfully using monoclonal antibody hybridoma tumour technology,which can be used to detect CL residues by competitive ELISA.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2004年第3期194-198,共5页
Chinese Journal of Immunology
基金
河南省科技攻关重大项目资助 (2 0 0 1招标 )
